Centromere DNA from yeast can be isolated, inserted into plasmids, and shown to exhibit the basic behavioral characteristics of intact centromeres in the genome. In these plasmids, centromere structure-function relationships are especially amenable to direct study. We will use this approach in the proposed research to investigate the molecular details of processes by which the centomere governs chromosome organization and movement in yeast. In previous work we have shown that two yeast centromere DNAs (CEN3 and CEN11) contain conserved arrangements of DNA sequence homology which are essential for centromere function. We will extend these observations by isolating and characterizing CEN7 from a very large yeast chromosome (VII), to examine structural similarities and differences. Using in vitro mutagenesis techniques to alter centromere plasmids, we will analyze the effect of specific sequence changes and structural rearrangements on centromere function. We will also study centromeric-flanking DNA segments that, as indicated by preliminary studies, may function to assure proper chromosome segregation and to maintain specific chromatin configurations. In addition, we will investigate a possible role for DNA methylation in centromere function. Yeast centromere DNA sequences are organized in a unique, nuclease-resistant chromatin core which may function as a primitive kinetochore. The single microtubule attachment site in yeast may, in fact, be a model repeat unit structure for the multiple attachment sites found in other, more complex, kinetochores.